The ability to reliably make large, perfect single crystals of various materials ranging from simple organic chemicals to complex macromolecules such as proteins and nucleic acids has long been an elusive goal in a wide variety of fields. The lengths which have been gone to in quest of that goal include setting up experiments which are designed to last weeks and even months, specially designed vibrationless rooms, complex equipment for countering convection currents and gravity, and even setting up experiments in the depths of outer space. There have been numerous attempts to solve this problem, which have met with varying degrees of success, but all of which suffer from drawbacks which limit their application widely, inexpensively, or reliably.
In order to make crystals, the material of interest is usually dissolved in a solvent and the solubility of the material is decreased, usually very gradually, in one of a number of ways, including increasing the salt concentration of the solution, evaporating off the solvent or cooling the solution. Alternatively, crystallization is induced by adding a seed crystal of purified material (if it is available) or by creating some other nucleus of crystallization. However, more often than not, either the material will not crystallize at all, i.e., it merely precipitates out of solution in an amorphous condition or simply dries, or, if crystals are formed, they are small, imperfect and/or not suitable for the purpose for which they were intended.
The range of utilities of single crystals is vast, and varies with the nature of the material as well as the size and purity. Crystals themselves may have utility, for example, as semiconductors and as other components used to process electrical or electromagnetic energy or optoelectronic signals. A very important utility is the study of crystals themselves as a tool for learning about the structure of the molecule of interest. Especially in the case of complex macromolecules, structural information is best achieved by X-ray diffraction studies, which require the use of single crystals. Protein single crystals, in particular, are essential for uncovering the three dimensional structure, or conformation, of the protein by X-ray crystallography. The entire field of protein engineering is built upon, and thus far limited to, knowledge obtained from X-ray crystallography. Protein engineering is the emerging science that enables one to alter protein and enzyme structures to impart desirable properties, useful to man but unobtainable naturally. For an enzyme (which is also a protein), the structure provides valuable information about why and how the enzyme works, and how one can improve it by protein engineering. Also, the three dimensional structural information is essential for drug design. A further utility of the process of this invention is the production of single crystal of zeolites, which may offer significantly improved selectivity in catalysis.
One problem of growing crystals, especially single protein crystals, is that there is no universally accepted standard method. Typically, different laboratories use different techniques and different source materials for the same protein. A coherent paradigm that provides a rational set of rules for protein crystal growth methods is acutely needed.
Protein crystallization is practiced commercially for three main purposes: purification, storage, and structure determination. For purification and storage, the crystals are relatively easy to grow and need not be large single crystals. Conversely, large single crystals are required for X-ray studies. In industrial applications for storage and purification, crystals do not need to be structurally pure, i.e., imperfections in their structures frequently do not interfere at all with economic value. Protein single crystals, however, must have very high degrees of structural purity for providing X-ray diffraction resolution adequate for accurate determination of three dimensional molecular network structure. For this reason, the strategy for making single crystals of proteins is to suppress nucleation, to provide only a few crystals and to create an environment for slow growth, to avoid imperfections from forming. In the laboratory the single crystals are generally grown either by cooling, evaporation, or dialysis. Inorganic salts, such as sodium chloride or ammonium sulfate, and polymers such as polyethylene glycol are almost always used to induce supersaturation (salting out).
These methods suffer from thermal and concentration gradients which cause convection currents that lead to imperfections in the crystals. The method of dialysis uses a membrane which allows salts to permeate to the protein solution, causing salting out. But this method does not apply to those proteins which require polyethylene glycol or other organic substances for the onset of nucleation. In the recent past, researchers, under the auspices of the National Aeronautics and Space Administration (NASA), have performed studies in microgravity with promising results. Whether microgravity can be a viable source for single crystals is doubtful, however, due to lack of access to long-duration space flights.
Therefore, the absence of a technology for growing large single crystals of proteins is a bottleneck in the progress of biotechnology. The pharmaceutical and the biotechnology industries in particular, as well as academic researchers, would benefit greatly from the development of a reliable method for making crystals, especially of proteins.